Therapeutical tools and methods for treating blindness

ABSTRACT

The present inventions relates to the use of an isolated nucleic acid molecule comprising a nucleotide sequence coding for a hyperpolarizing light-gated ion channel or pump gene from an archeon or for a light-active fragment of said gene, or the nucleotide sequence complementary to said nucleotide sequence, for treating or ameliorating blindness. The light-gated ion channel or pump gene can be a halorhodopsin gene.

FIELD OF THE INVENTION

The present invention relates to methods of treating blindness. Thepresent invention also relates to constructs for use in treatingblindness, as well as their use in the manufacture of a medicament fortreating blindness.

BACKGROUND OF THE INVENTION

Blindness is a major health problem that disables millions of peopleworldwide. The most common cause of blindness is the disfunction of theretina. The three most common forms of retinal blindness are retinitispigmentosa (RP), macular deneneration (MD) and glaucoma (G). In RP andMD the primary problem is the degeneration of photoreceptors and theconsequent loss of photosensitivity. There is thus a need to be able toobviate the problems associated with such degeneration ofphotoreceptors.

One approach has been to develop a retinal prosthesis, a “seeing eye”chip with as many as 1,000 tiny electrodes to be implanted in the eye.This would have the potential to help people who have lost their sightto regain enough vision to function independently, but the numbers ofelectrodes is simply insufficient to provide a high degree or level ofsight to be obtained. Moreover, there are problems associated withinserting foreign bodies into the eye. Recently a number of genes hasbeen isolated and/or manipulated that when expressed can make cellslight sensitive. In some cases additional non-genetic factors are alsoneeded to make cells light sensitive.

One proposal by Eli in 2001 was to use the chlorophyll-containingproteins in spinach to treat vision loss. These proteins give off asmall electrical voltage after capturing the energy of incoming photonsof light. Although, the research has shown that photosystem I reactioncentres can be incorporated into a liposome and are shown to befunctional, in that it produces the experimental equivalent of a voltagewhen light is shone on it, hitherto this has not been shown to work in aretinal cell.

Other work by neurobiologist Richard Kramer at UC Berkeley has looked atre-engineering a potassium channel to be responsive to light rather thanvoltage, in order to allow insertion of a light activated switch intobrain cells normally insensitive to light. However, the channel has tobe mutated so that it always stays open and a chemical “plug”, attachedto the channel, which is sensitive to light such that when lit withlong-wavelength UV light, the plug is released from the channel, lettingpotassium out of the channel. Light of a longer wavelength causes theplug to insert back into the channel and stop release of potassium. Itwill be appreciated however, that such a system is extremely complex andproblems are likely to arise if the channel is delivered to the wrongtype of retinal cells.

Bi et al., (Neuron, 50, 2006, p 23-33) discloses the use ofmicrobial-type rhodopsin to restore visual responses in mice withphotoreceptor degeneration. However, the expression of the rhodopsingene is likely to have occurred in a variety of types of cell in the eyewhich is potentially undesirable and/or problematic. It also appearsthat the threshold light intensity required for producing responses ismuch higher than for normal rod and cone photoreceptors, but there is noteaching of how this may be addressed in, for example, low lightenvironments.

An alternative method has been described by some of the presentinventors in WO-A-2008/022772, wherein e.g. channelrhodopsin-2 istargeted to e.g. ON-cells. This method has however the disadvantage ofbeing sub-optimal with OFF-cells.

It is amongst the objects of the present invention to obviate and/ormitigate at least one of the aforementioned disadvantages.

It is also an object of the present invention to provide a systemsuitable for use in preventing and/or treating blindness in a subject.

SUMMARY OF THE INVENTION

To address this need, the present inventors investigated the capacity ofa phylogenetically ancient hyperpolarizing light sensor, such as thehalorhodopsin from the archeon (archeabacteria) Natronomas pharaonis(NpHR), specifically expressed in rod and cone photoreceptors for itscapacity to restore vision in an experimental model for blindness.

Surprisingly, the inventors realized that this receptor on itself could,when introduced into rod and cone photoreceptors, stimulate both the ON-and the OFF-system, and restore some vision on itself, i.e. without theneed of any of the numerous other light transduction cascade componentsusually found in rod and cone photoreceptors

The present invention therefore encompasses an isolated nucleic acidmolecule comprising a nucleotide sequence coding for a hyperpolarizinglight-gated ion channel or pump gene from an archeon or for alight-active fragment of said gene, or the nucleotide sequencecomplementary to said nucleotide sequence, for use in treating orameliorating blindness. Said light-gated ion channel or pump gene can behalorhodopsin gene, for instance the halorhodopsin gene from Natronomaspharaonis (NpHR).

The isolated nucleic acid molecule of the invention can be used byadministration and expression in at least one of cones, rods, horizontalcells, rod bipolar cells, ON-cone bipolar cells, OFF-cone bipolar cells,amacrine cells, ganglion cells. Therefore, the isolated nucleic acidmolecule of the invention can comprise a cell specific promoter, forinstance human rhodopsin promoter, human red opsin promoter the Grm6promoter controlling the expression of the light-gated ion channel orpump gene.

Moreover, the isolated nucleic acid molecule of can be used,simultaneously or sequentially, together with a depolarizing light-gatedion channel gene, for example a channelrhodopsin, for instancechannelrhodopsin-2. Moreover, the depolarizing light-gated ion channelgene can be under the control of human rhodopsin promoter, human redopsin promoter and the Grm6 promoter.

The present invention also encompasses an isolated nucleic acid moleculeuseful for treating blindness comprising a nucleic acid sequence codingfor a hyperpolarizing light-gated ion channel or pump gene and a nucleicacid sequence coding for a depolarizing light-gated ion channel or pumpgene. An embodiment thereof is an isolated nucleic acid molecule whereinsaid hyperpolarizing light-gated ion channel or pump gene ishalorhodopsin, for instance the halorhodopsin gene from Natronomaspharaonis (NpHR), and said a depolarizing light-gated ion channel geneis a channelrhodopsin, for instance channelrhodopsin-2. In this case,the hyperpolarizing light-gated ion channel or pump and the depolarizinglight-gated ion channel can be encoded in such a way that a fusionprotein is formed upon expression. Moreover, both genes can be, eithercommonly or independently, under the control of a promoter chosen fromthe group of human rhodopsin promoter, human red opsin promoter and theGrm6 promoter.

The present invention further encompasses a recombinant vectorcomprising a nucleic acid of the invention or a host cell comprisingsaid vector.

In addition, the present invention also encompasses a kit comprising anisolated nucleic molecule of the invention, a recombinant vectorcomprising said molecule or a host cell comprising said vector.

DESCRIPTION OF THE FIGURES

FIG. 1: AAV2-Rho-NpHR-EYFP genomic vector

FIG. 2: Spectral tuning curve (mean total number of spikes during lightON normalized to each cell and light level) of AAV-NpHR-infected retinae(grey) and untreated control retinae (black) of mice (please note thatmice do not normally possess a red-light-sensitive receptor).

DETAILED DESCRIPTION OF THE INVENTION

To address this need, the present inventors investigated the capacity ofa phylogenetically ancient hyperpolarizing light sensor, such as thehalorhodopsin from the archeon (archeabacteria) Natronomas pharaonis(NpHR), specifically expressed in rod and cone photoreceptors for itscapacity to restore vision in an experimental model for blindness.Surprisingly, the inventors realized that this receptor on itself could,when introduced into rod and cone photoreceptors, stimulate both the ON-and the OFF-system, and restore some vision on itself, i.e. without theneed of any of the numerous other light transduction cascade componentsusually found in rod and cone photoreceptors

The present invention therefore encompasses an isolated nucleic acidmolecule comprising a nucleotide sequence coding for a hyperpolarizinglight-gated ion channel or pump gene from an archeon or for alight-active fragment of said gene, or the nucleotide sequencecomplementary to said nucleotide sequence, for use in treating orameliorating blindness. Said light-gated ion channel or pump gene can behalorhodopsin gene, for instance the halorhodopsin gene from Natronomaspharaonis (NpHR).

The isolated nucleic acid molecule of the invention can be used byadministration and expression in at least one of cones, rods, horizontalcells, rod bipolar cells, ON-cone bipolar cells, OFF-cone bipolar cells,amacrine cells, ganglion cells. Therefore, the isolated nucleic acidmolecule of the invention can comprise a cell specific promoter, forinstance human rhodopsin promoter, human red opsin promoter the Grm6promoter controlling the expression of the light-gated ion channel orpump gene.

Moreover, the isolated nucleic acid molecule of can be used,simultaneously or sequentially, together with a depolarizing light-gatedion channel gene, for example a channelrhodopsin, for instancechannelrhodopsin-2. Moreover, the depolarizing light-gated ion channelgene can be under the control of human rhodopsin promoter, human redopsin promoter and the Grm6 promoter.

The present invention also encompasses an isolated nucleic acid moleculeuseful for treating blindness comprising a nucleic acid sequence codingfor a hyperpolarizing light-gated ion channel or pump gene and a nucleicacid sequence coding for a depolarizing light-gated ion channel or pumpgene. An embodiment thereof is an isolated nucleic acid molecule whereinsaid hyperpolarizing light-gated ion channel or pump gene ishalorhodopsin, for instance the halorhodopsin gene from Natronomaspharaonis (NpHR), and said a depolarizing light-gated ion channel geneis a channelrhodopsin, for instance channelrhodopsin-2. In this case,the hyperpolarizing light-gated ion channel or pump and the depolarizinglight-gated ion channel can be encoded in such a way that a fusionprotein is formed upon expression. Moreover, both genes can be, eithercommonly or independently, under the control of a promoter chosen fromthe group of human rhodopsin promoter, human red opsin promoter and theGrm6 promoter.

The present invention further encompasses a recombinant vectorcomprising a nucleic acid of the invention or a host cell comprisingsaid vector.

In addition, the present invention also encompasses a kit comprising anisolated nucleic molecule of the invention, a recombinant vectorcomprising said molecule or a host cell comprising said vector.

Moreover, the present invention also encompasses method of treatingblindness using the isolated nucleic acid molecules or vectors of theinvention.

Compositions comprising the nucleic acid molecules of the invention arealso encompassed by the present invention. Said compositions can bepharmaceutically acceptable compositions.

Moreover, the nucleic acid molecules of the invention can be used tomanufacture medicaments and/or to treat patients. Hence, the presentinvention also encompasses methods of treatment using the nucleic acidmolecules of the invention

It is to be understood that the medicament is generally usedtherapeutically, but it may be used in a prophylactic sense, when asubject has been identified as being likely to suffer from blindness,but actual vision loss has not yet occurred or has only minimallyoccurred.

By “blindness” is meant total or partial loss of vision. Typically themedicament may be used to treat blindness associated with maculardegeneration, glaucoma and/or retinitis pigmentosa. However, it is to beappreciated that any disease or condition which leads to degeneration ornon-functioning of photoreceptors in the eye may be treated using themedicament. Moreover, without wishing to be bound by theory, it isbelieved that the present invention will be particularly effective forcuring blindness at early stages of retinal degeneration (rd) whenphotoreceptor function is lost but the photoreceptor-to-bipolar synapsemay still be intact.

An “active fragment of the light-gated ion channel or pump” is afragment which when expressed generates a polypeptide which is stillcapable of functioning as a light capturing molecule which causes asubsequent flow of ions into or out of the cell in which the channel islocated and a consequent change in voltage.

By “hyperpolarisation” is meant to decrease the membrane potential of acell. By “Depolarisation” is meant to increase the membrane potential ofa cell.

It will be appreciated that the present invention also extends tomethods of treating prophylactically or therapeutically blindness byadministering to a patient suffering or predisposed to developingblindness, a DNA construct according to the invention comprising alight-gated ion channel or pump gene sequence or active fragmentthereof, which gene sequence or fragment thereof is capable ofexpressing one or more copies of the light-gated ion channel orpumpprotein in a retinal cell, whereby expression of said one or morecopies of the light-gated ion channel or pump protein render the cellphotosensitive so as to enable treatment or amelioration of blindness.

Typically, the light-gated ion channel or pump gene sequence of theinvention or fragment thereof may be administered to a subject in theform of a recombinant molecule comprising said light-gated ion channelgene sequence or active fragment under appropriatetranscriptional/translational controls to allow expression of saidlight-gated ion channel or pump protein when administered to retinalcells of a subject. It will be appreciated that the light-gated ionchannel or pump sequence or fragment may be under control of a suitablepromoter, such as a constitutive and/or controllable promoter.

The present invention also therefore provides a recombinant molecule ofthe invention comprising a light-gated ion channel or pump gene sequenceor active fragment thereof for use in therapy. The recombinant moleculemay be in the form of a plasmid, phagemid or viral vector. Furthermore,recombinantly expressed, or chemically synthesised light-gated ionchannel or pump protein, or functionally important fragments thereof,may be produced and applied to the eye via a suitable ointment or otherpharmaceutical vehicle, as a treatment or prophylactic measure fortreating said aforementioned diseases.

Many different viral and non-viral vectors and methods of theirdelivery, for use in gene therapy, are known, such as adenovirusvectors, adeno-associated virus vectors, retrovirus vectors, lentiviralvectors, herpes virus vectors, liposomes, naked DNA administration andthe like. A detailed review of possible techniques for transforminggenes into desired cells of the eye is taught by Wright (Br JOphthalmol, 1997; 81: 620-622) which is incorporated herein byreference. Moreover, it may also be possible to use encapsulated celltechnology as developed by Neurotech, for example.

The light-gated ion channel or pump gene is a halorhodopsin gene and canbe if desired combined with a rhodopsin, such as a rhodopsin from amicroorganism, such as a unicellular alga, typically from the speciesChlamydononas, especially Chlamydomonas reinhardtii. A preferredrhodopsin is Channelrhodopsin-2 (ChR2) which is a light gated cationchannel from C. reinhardtii, see for example, Boyden et al 2005 (NatureNeuroscience, 8, 9; 1263-1268) and WO-A-2003/084994.

Preferably the cells to which the medicament or vector are to beadministered, and in which the gene is to be expressed are rod bipolarcells, ON cone bipolar cells, OFF cone bipolar cells, horizontal cells,amacrine cells and ganglion cells. Moreover, the photoreceptor cells(rod and cones) themselves which have lost photosensitivity, but whichare not “dead” can be used to express the light-gated ion channel orpump gene. Moreover expression of the light-gated ion channel or pumpgene in photoreceptors may serve to prevent or show down degeneration.

It is understood that it is preferable that expression of thelight-gated ion channel gene of the invention is controlled by way of acell specific promoter. Thus a cell specific promoter may be used toensure that the light-gated ion channel gene is only expressed in aspecific cell type. For example, the mGluR6 promoter (Ueda et al, JNeurosci. 1997 May 1; 17(9):3014-23) may be employed to controlexpression in ON-bipolar cells.

Once expressed in an appropriate retinal cell, the light-gated ionchannel or pump protein inserts within the plasma membrane of the cell,rendering the cell photosensitive and able to cause ion transport,cation or anion, in response to light. Nevertheless, although it isknown that the retina is sensitive to very wide ranges of lightintensities due to the adaptive nature of photoreceptors, light-gatedion channels or pumps may not be able to adapt and may therefore respondonly to a narrow range of light intensities. If this is the case, such alimitation may be mitigated by use of image intensifiers and/or imageconverters known in the art. For example, a patient who has been treatedby the above described method, may wear, image intensifiers/enhancersmounted, for example, on spectacles or the like.

By way of an example, an image intensifying device, such as thoseprovided by Telesensory (http://www.telesensory.com), may be combinedwith a retinal scanning device (RSD) as developed by Microvision(http://www.microvision.com/milprod.html), to provide a head-wornapparatus capable of delivering a bright, intensified image directly tothe retina of a patient with impaired vision(http://www.telesensory.com/home8.html). Briefly, a RSD projects imagesonto the retina such that an individual can view a large, full-motionimage without the need for additional screens or monitors. Thus, byprojecting an intensified image directly to the retina of an individualwith impaired vision, it may be possible to improve vision in thoseconsidered to be blind.

In case of expressing the light-gated ion channel or pump in retinalbipolar or ganglion cells some aspects of the network processingcapabilities of the retina can be lost. For example horizontal cellmediated lateral inhibition can be lost if light activates bipolar organglion cells. In these cases a retina like processor (D. Balya and B.Roska: “Retina model with real time implementation”, InternationalSymposium on Circuits and Systems ISCAS 2005, Kobe, Japan, May, pp.5222-5225, also see http://www.anafocus.com/and http://www.eutecus.com/)can be combined with the Microvision system.

If the light-gated ion channel or pump is expressed in photoreceptors asmentioned before the polarity of light response in photoreceptors caninverse. That can be corrected with inverting the polarity of theprojected image: dark pixels becoming light and light pixels becomingdark.

These and other aspects of the present invention should be apparent tothose skilled in the art, from the teachings herein.

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are also providedbelow.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The “Archaea” are a group of prokaryotic and single-celledmicroorganisms. In this they are similar to bacteria but these twogroups evolved differently, and are classified as different domains inthe three-domain system. Originally these organisms were namedarchaebacteria. Although there is still uncertainty in the phylogeny,Archaea, Eukaryota and Bacteria were introduced as the fundamentalclassifications in the three-domain system by Carl Woese in 1977. Asprokaryotes, archaea are also classified in kingdom Monera in thetraditional five-kingdom Linnaean taxonomy. While their prokaryotic cellstructure is similar to bacteria, the genes of Archaea and several oftheir metabolic pathways are more closely related to those ofeukaryotes. One way to account for this is to group archaeans andeukaryotes together in the clade Neomura, which might have arisen fromgram-positive bacteria. On the other hand, other studies have suggestedthat Archaea may instead be the most ancient lineage in the world, withbacteria and eukaryotes diverging from this group. Archaea wereoriginally described in extreme environments, but have since been foundin all habitats and may contribute up to 20% of total biomass. Thesecells are particularly common in the oceans, and the archaea in planktonmay be one of the most abundant groups of organisms on the planet. Asingle individual or species from this domain is called an archaeon(sometimes spelled “archeon”) while the adjectival form is archaeal orarchaean.

“Halorhodopsin” is a light-driven ion pump, specific for chloride ions,and found in phylogenetically ancient “bacteria” (archaea), known ashalobacteria. It is a seven-transmembrane protein of the retinylideneprotein family, homologous to the light-driven proton pumpbacteriorhodopsin, and similar in tertiary structure (but not primarysequence structure) to vertebrate rhodopsins, the pigments that senselight in the retina. Halorhodopsin also shares sequence similarity tochannelrhodopsin, a light-driven ion channel. Halorhodopsin contains theessential light-isomerizable vitamin A derivative all-trans-retinal.Halorhodopsin is one of the few membrane proteins whose crystalstructure is known. Halorhodopsin isoforms can be found in multiplespecies of halobacteria, including H. salinarum, and N. pharaonis. Muchongoing research is exploring these differences, and using them to parseapart the photocycle and pump properties. After bacteriorhodopsin,halorhodopsin may be the best type I (microbial) opsin studied. Peakabsorbance of the halorhodopsin retinal complex is about 570 nm.Recently, halorhodopsin has become a tool in optogenetics. Just as theblue-light activated ion channel channelrhodopsin-2 opens up the abilityto activate excitable cells (such as neurons, muscle cells, pancreaticcells, and immune cells) with brief pulses of blue light, halorhodopsinopens up the ability to silence excitable cells with brief pulses ofyellow light. Thus halorhodopsin and channelrhodopsin together enablemultiple-color optical activation, silencing, and desynchronization ofneural activity, creating a powerful neuroengineering toolbox.

“Polynucleotide” and “nucleic acid”, used interchangeably herein, referto polymeric forms of nucleotides of any length, either ribonucleotidesor deoxyribonucleotides. Thus, these terms include, but are not limitedto, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. These terms further include, but are notlimited to, mRNA or cDNA that comprise intronic sequences. The backboneof the polynucleotide can comprise sugars and phosphate groups (as maytypically be found in RNA or DNA), or modified or substituted sugar orphosphate groups. Alternatively, the backbone of the polynucleotide cancomprise a polymer of synthetic subunits such as phosphoramidites andthus can be an oligodeoxynucleoside phosphoramidate or a mixedphosphoramidate-phosphodiester oligomer. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs, uracyl, other sugars, and linking groups such as fluororiboseand thioate, and nucleotide branches. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications included in thisdefinition are caps, substitution of one or more of the naturallyoccurring nucleotides with an analog, and introduction of means forattaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support. The term“polynucleotide” also encompasses peptidic nucleic acids, PNA and LNA.Polynucleotides may further comprise genomic DNA, cDNA, or DNA-RNAhybrids.

“Sequence Identity” refers to a degree of similarity or complementarity.There may be partial identity or complete identity. A partiallycomplementary sequence is one that at least partially inhibits anidentical sequence from hybridizing to a target polynucleotide; it isreferred to using the functional term “substantially identical.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially identical sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely identical sequence or probe to the target sequence underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarities (e.g.,less than about 30% identity); in the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Another way of viewing sequence identity in the context to two nucleicacid or polypeptide sequences includes reference to residues in the twosequences that are the same when aligned for maximum correspondence overa specified region. As used herein, percentage of sequence identitymeans the value determined by comparing two optimally aligned sequencesover a comparison window, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

“Gene” refers to a polynucleotide sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence. A gene may constitutean uninterrupted coding sequence or it may include one or more introns,bound by the appropriate splice junctions. Moreover, a gene may containone or more modifications in either the coding or the untranslatedregions that could affect the biological activity or the chemicalstructure of the expression product, the rate of expression, or themanner of expression control. Such modifications include, but are notlimited to, mutations, insertions, deletions, and substitutions of oneor more nucleotides. In this regard, such modified genes may be referredto as “variants” of the “native” gene.

“Expression” generally refers to the process by which a polynucleotidesequence undergoes successful transcription and translation such thatdetectable levels of the amino acid sequence or protein are expressed.In certain contexts herein, expression refers to the production of mRNA.In other contexts, expression refers to the production of protein.

“Cell type” refers to a cell from a given source (e.g., tissue or organ)or a cell in a given state of differentiation, or a cell associated witha given pathology or genetic makeup.

“Polypeptide” and “protein”, used interchangeably herein, refer to apolymeric form of amino acids of any length, which may includetranslated, untranslated, chemically modified, biochemically modified,and derivatized amino acids. A polypeptide or protein may be naturallyoccurring, recombinant, or synthetic, or any combination of these.Moreover, a polypeptide or protein may comprise a fragment of anaturally occurring protein or peptide. A polypeptide or protein may bea single molecule or may be a multi-molecular complex. In addition, suchpolypeptides or proteins may have modified peptide backbones. The termsinclude fusion proteins, including fusion proteins with a heterologousamino acid sequence, fusions with heterologous and homologous leadersequences, with or without N-terminal methionine residues,immunologically tagged proteins, and the like.

A “fragment of a protein” refers to a protein that is a portion ofanother protein. For example, fragments of proteins may comprisepolypeptides obtained by digesting full-length protein isolated fromcultured cells. In one embodiment, a protein fragment comprises at leastabout 6 amino acids. In another embodiment, the fragment comprises atleast about 10 amino acids. In yet another embodiment, the proteinfragment comprises at least about 16 amino acids.

An “expression product” or “gene product” is a biomolecule, such as aprotein or mRNA, that is produced when a gene in an organism istranscribed or translated or post-translationally modified.

“Host cell” refers to a microorganism, a prokaryotic cell, a eukaryoticcell or cell line cultured as a unicellular entity that may be, or hasbeen, used as a recipient for a recombinant vector or other transfer ofpolynucleotides, and includes the progeny of the original cell that hasbeen transfected. The progeny of a single cell may not necessarily becompletely identical in morphology or in genomic or total DNA complementas the original parent due to natural, accidental, or deliberatemutation.

The term “functional equivalent” is intended to include the “fragments”,“mutants”, “derivatives”, “alleles”, “hybrids”, “variants”, “analogs”,or “chemical derivatives” of the native gene or virus.

“Isolated” refers to a polynucleotide, a polypeptide, an immunoglobulin,a virus or a host cell that is in an environment different from that inwhich the polynucleotide, the polypeptide, the immunoglobulin, the virusor the host cell naturally occurs.

“Substantially purified” refers to a compound that is removed from itsnatural environment and is at least about 60% free, at least about 65%free, at least about 70% free, at least about 75% free, at least about80% free, at least about 83% free, at least about 85% free, at leastabout 88% free, at least about 90% free, at least about 91% free, atleast about 92% free, at least about 93% free, at least about 94% free,at least about 95% free, at least about 96% free, at least about 97%free, at least about 98% free, at least about 99% free, at least about99.9% free, or at least about 99.99% or more free from other componentswith which it is naturally associated.

“Diagnosis” and “diagnosing” generally includes a determination of asubject's susceptibility to a disease or disorder, a determination as towhether a subject is presently affected by a disease or disorder, aprognosis of a subject affected by a disease or disorder (e.g.,identification of pre-metastatic or metastatic cancerous states, stagesof cancer, or responsiveness of cancer to therapy), and therametrics(e.g., monitoring a subject's condition to provide information as to theeffect or efficacy of therapy).

“Biological sample” encompasses a variety of sample types obtained froman organism that may be used in a diagnostic or monitoring assay. Theterm encompasses blood and other liquid samples of biological origin,solid tissue samples, such as a biopsy specimen, or tissue cultures orcells derived therefrom and the progeny thereof. The term specificallyencompasses a clinical sample, and further includes cells in cellculture, cell supernatants, cell lysates, serum, plasma, urine, amnioticfluid, biological fluids, and tissue samples. The term also encompassessamples that have been manipulated in any way after procurement, such astreatment with reagents, solubilization, or enrichment for certaincomponents.

“Individual”, “subject”, “host” and “patient”, used interchangeablyherein, refer to any mammalian subject for whom diagnosis, treatment, ortherapy is desired. In one preferred embodiment, the individual,subject, host, or patient is a human. Other subjects may include, butare not limited to, cattle, horses, dogs, cats, guinea pigs, rabbits,rats, primates, and mice.

“Hybridization” refers to any process by which a polynucleotide sequencebinds to a complementary sequence through base pairing. Hybridizationconditions can be defined by, for example, the concentrations of salt orformamide in the prehybridization and hybridization solutions, or by thehybridization temperature, and are well known in the art. Hybridizationcan occur under conditions of various stringency.

“Stringent conditions” refers to conditions under which a probe mayhybridize to its target polynucleotide sequence, but to no othersequences. Stringent conditions are sequence-dependent (e. g., longersequences hybridize specifically at higher temperatures). Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionicstrength, pH, and polynucleotide concentration) at which 50% of theprobes complementary to the target sequence hybridize to the targetsequence at equilibrium. Typically, stringent conditions will be thosein which the salt concentration is at least about 0.01 to about 1.0 Msodium ion concentration (or other salts) at about pH 7.0 to about pH8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides).

Stringent conditions may also be achieved with the addition ofdestabilizing agents, such as formamide.

“Biomolecule” includes polynucleotides and polypeptides.

“Biological activity” refers to the biological behavior and effects of aprotein or peptide. The biological activity of a protein may be affectedat the cellular level and the molecular level. For example, thebiological activity of a protein may be affected by changes at themolecular level. For example, an antisense oligonucleotide may preventtranslation of a particular mRNA, thereby inhibiting the biologicalactivity of the protein encoded by the mRNA. In addition, animmunoglobulin may bind to a particular protein and inhibit thatprotein's biological activity.

“Oligonucleotide” refers to a polynucleotide sequence comprising, forexample, from about 10 nucleotides (nt) to about 1000 nt.Oligonucleotides for use in the invention are for instance from about 15nt to about 150 nt, for instance from about 150 nt to about 1000 nt inlength. The oligonucleotide may be a naturally occurring oligonucleotideor a synthetic oligonucleotide.

“Modified oligonucleotide” and “Modified polynucleotide” refer tooligonucleotides or polynucleotides with one or more chemicalmodifications at the molecular level of the natural molecular structuresof all or any of the bases, sugar moieties, internucleoside phosphatelinkages, as well as to molecules having added substitutions or acombination of modifications at these sites. The internucleosidephosphate linkages may be phosphodiester, phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate,bridged phosphorothioate or sulfone internucleotide linkages, or 3′-3′,5′-3′, or 5′-5′ linkages, and combinations of such similar linkages. Thephosphodiester linkage may be replaced with a substitute linkage, suchas phosphorothioate, methylamino, methylphosphonate, phosphoramidate,and guanidine, and the ribose subunit of the polynucleotides may also besubstituted (e. g., hexose phosphodiester; peptide nucleic acids). Themodifications may be internal (single or repeated) or at the end (s) ofthe oligonucleotide molecule, and may include additions to the moleculeof the internucleoside phosphate linkages, such as deoxyribose andphosphate modifications which cleave or crosslink to the opposite chainsor to associated enzymes or other proteins. The terms “modifiedoligonucleotides” and “modified polynucleotides” also includeoligonucleotides or polynucleotides comprising modifications to thesugar moieties (e. g., 3′-substituted ribonucleotides ordeoxyribonucleotide monomers), any of which are bound together via 5′ to3′ linkages.

“Biomolecular sequence” or “sequence” refers to all or a portion of apolynucleotide or polypeptide sequence.

The term “detectable” refers to a polynucleotide expression patternwhich is detectable via the standard techniques of polymerase chainreaction (PCR), reverse transcriptase-(RT) PCR, differential display,and Northern analyses, which are well known to those of skill in theart. Similarly, polypeptide expression patterns may be “detected” viastandard techniques including immunoassays such as Western blots.

A “target gene” refers to a polynucleotide, often derived from abiological sample, to which an oligonucleotide probe is designed tospecifically hybridize. It is either the presence or absence of thetarget polynucleotide that is to be detected, or the amount of thetarget polynucleotide that is to be quantified. The targetpolynucleotide has a sequence that is complementary to thepolynucleotide sequence of the corresponding probe directed to thetarget. The target polynucleotide may also refer to the specificsubsequence of a larger polynucleotide to which the probe is directed orto the overall sequence (e.g., gene or mRNA) whose expression level itis desired to detect.

A “target protein” refers to a polypeptide, often derived from abiological sample, to which a protein-capture agent specificallyhybridizes or binds. It is either the presence or absence of the targetprotein that is to be detected, or the amount of the target protein thatis to be quantified. The target protein has a structure that isrecognized by the corresponding protein-capture agent directed to thetarget. The target protein or amino acid may also refer to the specificsubstructure of a larger protein to which the protein-capture agent isdirected or to the overall structure (e. g., gene or mRNA) whoseexpression level it is desired to detect.

“Complementary” refers to the topological compatibility or matchingtogether of the interacting surfaces of a probe molecule and its target.The target and its probe can be described as complementary, andfurthermore, the contact surface characteristics are complementary toeach other. Hybridization or base pairing between nucleotides or nucleicacids, such as, for example, between the two strands of adouble-stranded DNA molecule or between an oligonucleotide probe and atarget are complementary.

“Label” refers to agents that are capable of providing a detectablesignal, either directly or through interaction with one or moreadditional members of a signal producing system. Labels that aredirectly detectable and may find use in the invention includefluorescent labels. Specific fluorophores include fluorescein,rhodamine, BODIPY, cyanine dyes and the like.

The term “fusion protein” refers to a protein composed of two or morepolypeptides that, although typically not joined in their native state,are joined by their respective amino and carboxyl termini through apeptide linkage to form a single continuous polypeptide. It isunderstood that the two or more polypeptide components can either bedirectly joined or indirectly joined through a peptide linker/spacer.

The term “normal physiological conditions” means conditions that aretypical inside a living organism or a cell. Although some organs ororganisms provide extreme conditions, the intra-organismal andintra-cellular environment normally varies around pH 7 (i.e., from pH6.5 to pH 7.5), contains water as the predominant solvent, and exists ata temperature above 0° C. and below 50° C. The concentration of varioussalts depends on the organ, organism, cell, or cellular compartment usedas a reference.

“BLAST” refers to Basic Local Alignment Search Tool, a technique fordetecting ungapped sub-sequences that match a given query sequence.

“BLASTP” is a BLAST program that compares an amino acid query sequenceagainst a protein sequence database. “BLASTX” is a BLAST program thatcompares the six-frame conceptual translation products of a nucleotidequery sequence (both strands) against a protein sequence database.

A “cds” is used in a Gen Bank DNA sequence entry to refer to the codingsequence. A coding sequence is a sub-sequence of a DNA sequence that issurmised to encode a gene.

A “consensus” or “contig sequence”, as understood herein, is a group ofassembled overlapping sequences, particularly between sequences in oneor more of the databases of the invention.

The nucleic acid molecules of the present invention can be produced by avirus harbouring a nucleic acid that encodes the relevant gene sequence.The virus may comprise elements capable of controlling and/or enhancingexpression of the nucleic acid. The virus may be a recombinant virus.The recombinant virus may also include other functional elements. Forinstance, recombinant viruses can be designed such that the viruses willautonomously replicate in the target cell. In this case, elements thatinduce nucleic acid replication may be required in a recombinant virus.The recombinant virus may also comprise a promoter or regulator orenhancer to control expression of the nucleic acid as required. Tissuespecific promoter/enhancer elements may be used to regulate expressionof the nucleic acid in specific cell types. The promoter may beconstitutive or inducible.

A “promoters” is a region of DNA that is generally located upstream(towards the 5′ region) of the gene that is needed to be transcribed.The promoter permits the proper activation or repression of the genewhich it controls. Examples of promoters which are suitable for theinvention are the human rhodopsin promoter (Allocca et al., Novel AAVserotypes efficiently transduce murine photoreceptors, J Virol. (2007)),the human red opsin promoter (Nathan et al., Science. 1986 Apr. 11;232(4747):193-202), the red cone opsin promoter, the arr3 promoter (Zhu,X. et al. Mouse cone arrestin gene characterization: promoter targetsexpression to cone photoreceptors. FEBS Letters 524, 116-122 (2002)) orthe Grm6 promoter (Masu, M. et al. Specific deficit of the ON responsein visual transmission by targeted disruption of the mGluR6 gene. Cell80, 757-765 (1995)).

Contaminant components of its natural environment are materials thatwould interfere with the methods and compositions of the invention, andmay include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. Ordinarily, an isolated agent will be preparedby at least one purification step. In one embodiment, the agent ispurified to at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 88%, at least about 90%, at least about 92%, at least about 95%,at least about 97%, at least about 98%, at least about 99%, at leastabout 99.9%, or at least about 99.99% by weight.

“Expressing” a protein in a cell means to ensure that the protein ispresent in the cell, e. g., for the purposes of a procedure of interest.In numerous embodiments, “expressing” a protein will compriseintroducing a transgene into a cell comprising a polynucleotide encodingthe protein, operably linked to a promoter, wherein the promoter is aconstitutive promoter, or an inducible promoter where the conditionssufficient for induction are created, as well as a localizationsequence. However, a cell that, e. g., naturally expresses a protein ofinterest, can be used without manipulation and is considered as“expressing” the protein.

A “fluorescent probe” refers to any compound with the ability to emitlight of a certain wavelength when activated by light of anotherwavelength.

“Fluorescence” refers to any detectable characteristic of a fluorescentsignal, including intensity, spectrum, wavelength, intracellulardistribution, etc.

“Detecting” fluorescence refers to assessing the fluorescence of a cellusing qualitative or quantitative methods. For instance, thefluorescence is determined using quantitative means, e. g., measuringthe fluorescence intensity, spectrum, or intracellular distribution,allowing the statistical comparison of values obtained under differentconditions. The level can also be determined using qualitative methods,such as the visual analysis and comparison by a human of multiplesamples, e. g., samples detected using a fluorescent microscope or otheroptical detector (e. g., image analysis system, etc.) An “alteration” or“modulation” in fluorescence refers to any detectable difference in theintensity, intracellular distribution, spectrum, wavelength, or otheraspect of fluorescence under a particular condition as compared toanother condition. For example, an “alteration” or “modulation” isdetected quantitatively, and the difference is a statisticallysignificant difference. Any “alterations” or “modulations” influorescence can be detected using standard instrumentation, such as afluorescent microscope, CCD, or any other fluorescent detector, and canbe detected using an automated system, such as the integrated systems,or can reflect a subjective detection of an alteration by a humanobserver.

An assay performed in a “homogeneous format” means that the assay can beperformed in a single container, with no manipulation or purification ofany components being required to determine the result of the assay, e.g., a test agent can be added to an assay system and any effectsdirectly measured. Often, such “homogeneous format” assays will compriseat least one component that is “quenched” or otherwise modified in thepresence or absence of a test agent. ell.

Any of a number of cell types can be used in the present invention. Forexample, any eukaryotic cell, including plant, animal, and fungal cellscan be used. In some embodiments, neurone will be used. As used herein,“cells” can include whole cells (untreated cells), permeabilized cells,isolated mitochondria, and proteoliposomes, e. g., proteoliposomesreconstituted with a UCP or another protein of interest. The care andmaintenance of cells, including yeast cells, is well known to those ofskill in the art and can be found in any of a variety of sources, suchas Freshney (1994) Culture of Animal Cells. Manual of Basic Technique,Wiley-Liss, New York, Guthrie & Fink (1991), Guthrie and Fink, Guide toYeast Genetics and Molecular Biology, Academic Press, Ausubel et al.(1999) Current Protocols in Molecular Biology, Greene PublishingAssociates, and others.

Cells can be used at any of a wide range of densities, depending on thedye, the test agent, and the particular assay conditions. For instance,a density of about OD₆₀₀=0.01 to 1 is used, for example between about0.05 and 0.5, e.g. about 0.1.

Methods for expressing heterologous proteins in cells are well known tothose of skill in the art, and are described, e. g., in Ausubel (1999),Guthrie and Fink (1991), Sherman, et al. (1982) Vlethods ineastGenetics, Cold Spring Harbor Laboratories, Freshney, and others.Typically, in such embodiments, a polynucleotide encoding a heterologousprotein of interest will be operably linked to an appropriate expressioncontrol sequence for the particular host cell in which the heterologousprotein is to be expressed. Any of a large number of well-knownpromoters can be used in such method. The choice of the promoter willdepend on the expression levels to be achieved and on the desiredcellular specificity. Additional elements such as polyadenylationsignals, 5′ and 3′ untranslated sequences, etc. are also described inwell-known reference books.

In metazoan (animals having the body composed of cells differentiatedinto tissues and organs) cells, promoters and other elements forexpressing heterologous proteins are commonly used and are well known tothose of skill. See, e. g., Cruz & Patterson (1973) Tissue Culture,Academic Press; Meth. Enzymology 68 (1979), Academic Press; Freshney,3rd Edition (1994) Culture of Animal Cells: A Manual of BasicTechniques, Wiley-Liss. Promoters and control sequences for such cellsinclude, e. g., the commonly used early and late promoters from SimianVirus 40 (SV40), or other viral promoters such as those from polyoma,adenovirus 2, bovine papilloma virus, or avian sarcoma viruses, herpesvirus family (e. g., cytomegalovirus, herpes simplex virus, orEpstein-Barr Virus), or immunoglobulin promoters and heat shockpromoters (see, e. g. Sambrook, Ausubel, Meth. Enzymology Pouwells, etal., supra (1987)). In addition, regulated promoters, such asmetallothionein, (i. e., MT-1 and MT-2), glucocorticoid, or antibioticgene “switches” can be used. Enhancer regions of such promoters can alsobe used.

Expression cassettes are typically introduced into a vector thatfacilitates entry of the expression cassette into a host cell andmaintenance of the expression cassette in the host cell. Such vectorsare commonly used and are well know to those of skill in the art.Numerous such vectors are commercially available, e. g., fromInvitrogen, Stratagene, Clontech, etc., and are described in numerousguides, such as Ausubel, Guthrie, Strathem, or Berger, all supra. Suchvectors typically include promoters, polyadenylation signals, etc. inconjunction with multiple cloning sites, as well as additional elementssuch as origins of replication, selectable marker genes (e. g., LEU2,URA3, TRP 1, HIS3, GFP), centromeric sequences, etc.

For expression in mammalian cells, any of a number of vectors can beused, such as pSV2, pBC12BI, and p91023, as well as lytic virus vectors(e. g., vaccinia virus, adenovirus, baculovirus), episomal virus vectors(e. g., bovine papillomavirus), and retroviral vectors (e. g., murineretroviruses).

As used herein, the term “disorder” refers to an ailment, disease,illness, clinical condition, or pathological condition.

As used herein, the term “pharmaceutically acceptable carrier” refers toa carrier medium that does not interfere with the effectiveness of thebiological activity of the active ingredient, is chemically inert, andis not toxic to the patient to whom it is administered.

As used herein, the term “pharmaceutically acceptable derivative” refersto any homolog, analog, or fragment of an agent, e.g. identified using amethod of screening of the invention, that is relatively non-toxic tothe subject.

The term “therapeutic agent” refers to any molecule, compound, ortreatment, that assists in the prevention or treatment of disorders, orcomplications of disorders.

Compositions comprising such an agent formulated in a compatiblepharmaceutical carrier may be prepared, packaged, and labeled fortreatment.

If the complex is water-soluble, then it may be formulated in anappropriate buffer, for example, phosphate buffered saline or otherphysiologically compatible solutions.

Alternatively, if the resulting complex has poor solubility in aqueoussolvents, then it may be formulated with a non-ionic surfactant such asTween, or polyethylene glycol. Thus, the compounds and theirphysiologically acceptable solvates may be formulated for administrationby inhalation or insufflation (either through the mouth or the nose) ororal, buccal, parenteral, rectal administration or, in the case oftumors, directly injected into a solid tumor.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e. g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e. g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e. g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e. g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e. g., magnesium stearate, talc or silica); disintegrants(e. g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e. g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e. g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e. g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e. g.,in ampoules or in multi-dose containers, with an added preservative.

The compositions may take such forms as suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e. g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e. g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

The compounds may also be formulated as a topical application, such as acream or lotion.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection.

Thus, for example, the compounds may be formulated with suitablepolymeric or hydrophobic materials (for example, as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt. Liposomes andemulsions are well known examples of delivery vehicles or carriers forhydrophilic drugs.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically or prophylactically effective amounts of thecompositions in pharmaceutically acceptable form.

The composition in a vial of a kit may be in the form of apharmaceutically acceptable solution, e. g., in combination with sterilesaline, dextrose solution, or buffered solution, or otherpharmaceutically acceptable sterile fluid. Alternatively, the complexmay be lyophilized or desiccated; in this instance, the kit optionallyfurther comprises in a container a pharmaceutically acceptable solution(e. g., saline, dextrose solution, etc.), preferably sterile, toreconstitute the complex to form a solution for injection purposes.

In another embodiment, a kit further comprises a needle or syringe,preferably packaged in sterile form, for injecting the complex, and/or apackaged alcohol pad. Instructions are optionally included foradministration of compositions by a clinician or by the patient.

In one embodiment, an isolated nucleic acid molecule comprising anucleotide sequence coding for a hyperpolarizing light-gated ion channelor pump gene from an archeon, or for a light-active fragment of saidgene, will be expressed, eventually together with a channelrohdopsin,e.g. channelrhodpsin-2 (ChR2), exclusively in ON-bipolar cells, whereasa cation-conducting channelrhodopsin, e.g. the vChR1 derived from Volvoxcarteri (Zhang et al., Red-shifted optogenetic excitation: a tool forfast neural control derived from Volvox carteri, Nat Neurosci. 2008 Apr.23; doi:10.1038/nn.2120) will be expressed exclusively in OFF bipolarcells. Without wishing to be bound by theory, it is believed that thistargeted expression of three neuromodulators will result in anexcitation of the ON pathway by blue light (ChR2 component), andinhibition of said ON pathway by red light (NpHR component) whereas theOFF pathway will be excited by red light (vChR1 component). Hence, bluelight would mimic light ON and red light would mimic light OFF. Theadvantage of this system is believed to be that the OFF bipolar cellswould be directly excited (meaning depolarized) by vChR1. Withoutwishing to be bound by theory, it is believed that this combined andtargeted expression of e.g. ChR2, NpHR and vChR1 might improve thespectrum of artificial photoreceptors after photoreceptor loss inquantity (more and different bipolar cells, ON and OFF) and mightestablish bichromatic vision (blue=white and red=black).

In addition, it is believed that the combination of ChR2/NpHR in ONbipolar cells might help to establish a sharp boundary between these twotypes of artificial photoreceptors since it could shut down the ONsignal when red light is on and the OFF pathway will be turned on. Asexplained herein, the Grm6 promoter (Masu, M. et al. Specific deficit ofthe ON response in visual transmission by targeted disruption of themGluR6 gene. Cell 80, 757-765 (1995).) is an exemplary promoter thatcould be used to drive expression of ChR2/NpHR in ON bipolar cells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES

Genetic Approaches:

The present inventors expressed the red-light-sensitive chloride pumpNpHR (Zhang et al., Multimodal fast optical interrogation of neuralcircuitry, Nature Vo1466 (2007)) in photoreceptors of C57BL/6 mice usingadeno associated virus vector, AAV2/7 (Allocca et al., Novel AAVserotypes efficiently transduce murine photoreceptors, J Virol. (2007)),mediated gene transfer. NpHR was driven from the human rhodopsinpromoter.

Detection of NpHR in Infected Retinae:

AAV2/7-Rho-NpHR-EYFP (5.35E+12 particles/ml) was injected subretinallyinto adult C57BL/6 mice. Retinae of infected animals were prepared atdifferent time points post injection. These retinae wereantibody-stained for EYFP to label NpHR-expressing cells and with DAPIto visualize all cells in the outer nuclear layer (ONL). Confocalmicroscopy recordings were performed and these were analyzed by usingBitplane's Imaris (5.7.2) software.

The quantification of infected photoreceptors was done by countingNpHR-expressing cells relative to DAPI-labeled cells in confocal slices.

Multielectrode Array Recordings of AAV-Infected Retinas:

The photoreceptors of AAV-infected retinae were stimulated by usinglight of defined wavelengths and intensities. The spiking output ofganglion cells was recorded by MEA. The spectral tuning curve of AAVinfected retinas showed significantly higher light sensitivity at longerwavelengths compared to control untreated retinas (FIG. 2).

Results:

With these experiments, the present inventors were able to show thatAAV2/7 in combination with the human rhodopsin promoter is an excellenttool for the delivery of NpHR into photoreceptors. The onset of NpHRexpression was very fast (6 days post injection) and stable over a longperiod. As reported before, AAV2/7 particles penetrate the whole outernuclear layer independent of injection site and ONL depth (Li et al.,Cone-specific expression using a human red opsin promoter in recombinantAAV, Vision Research 48 (2008)). AAV infections at PO were lesseffective and likely rod restricted. The red-light-sensitive chloridepump NpHR was found to be functional in retinal explants. The obtainedresults demonstrate that NpHR can modulate photoreceptor activitytowards higher wavelengths (Jacobs et al., Emergence of novel colorvision in mice engineered to express a human cone photopigment, Science,VOL 315 (2007)).

The invention claimed is:
 1. An isolated nucleic acid moleculecomprising a nucleotide sequence coding in a 5′ to 3′ orientation for:(a) a human rhodopsin promoter or human red opsin promoter; and (b) aNatronomas pharaonis (NpHR) hyperpolarizing light-gated ion channel orpump gene or a light-active fragment of the gene.
 2. The isolatednucleic acid molecule of claim 1 further comprising: (c) a nucleotidesequence coding for a depolarizing light-gated ion channel or pump gene.3. The isolated nucleic acid molecule of claim 2, wherein thedepolarizing light-gated ion channel or pump gene is a channelrhodopsin.4. The isolated nucleic acid molecule of claim 2, wherein thehyperpolarizing light-gated ion channel or pump and the depolarizinglight-gated ion channel or pump are encoded to form a fusion protein. 5.The isolated nucleic acid molecule of claim 2, wherein expression of thegene encoding the depolarizing light-gated ion channel or pump is underthe control of a human rhodopsin promoter or human red opsin promoter.6. A recombinant vector comprising the nucleic acid molecule of claim 1.7. An isolated host cell comprising the vector of claim
 6. 8. Theisolated nucleic acid molecule of claim 1, wherein the promoter is ahuman rhodopsin promoter.
 9. The isolated nucleic acid molecule of claim1, wherein the promoter is a human red opsin promoter.
 10. The isolatednucleic acid molecule of claim 3, wherein the channelrhodopsin ischannelrhodopsin-2.
 11. A method of treating or ameliorating blindnessin a subject, the method comprising: administering to a retinal cell ofthe subject an isolated nucleic acid molecule comprising a nucleotidesequence coding in a 5′ to 3′ orientation for: (a) a human rhodopsinpromoter or human red opsin promoter operably linked to; and (b) aNatronomas pharaonis (NpHR) hyperpolarizing light-gated ion channel orpump gene or a light-active fragment of the gene, thereby treating orameliorating blindness in the subject.
 12. The method of claim 11,wherein the retinal cell is a cone cell.
 13. The method of claim 11,wherein the retinal cell is a rod cell.
 14. The method of claim 11further comprising simultaneously or sequentially administering adepolarizing light-gated ion channel or pump gene.
 15. The method ofclaim 14, wherein the depolarizing light-gated ion channel or pump geneis a channelrhodopsin.
 16. The method of claim 15, wherein thechannelrhodopsin is channelrhodopsin-2.
 17. The method of claim 14,wherein the depolarizing light-gated ion channel or pump gene is underthe control of a human rhodopsin promoter.
 18. The method of claim 14,wherein the depolarizing light-gated ion channel or pump gene is underthe control of a human red opsin promoter.